Methods and compositions for upregulating endogenous antioxidant systems

ABSTRACT

A nutritional supplement for reducing free radical damage is disclosed. The nutritional supplement includes a first vehicle comprising an upregulating compound mixture configured to upregulate an endogenous antioxidant system and an exogenous antioxidant mixture and a second vehicle comprising a mineral mixture. The upregulating compound mixture is configured to upregulate an endogenous antioxidant system to protect against free radical damage. The upregulating compound mixture includes alpha lipoic acid, resveratrol, curcumin, EGCG, Olivol®, rutin, quercetin, and hesperetin. The endogenous antioxidant system includes transcription factors such as Nrf2, NF-κB, PPARα, PPARβ/δ, and PPARγ that promote transcription of antioxidant genes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/359,106 (Attorney Docket No. 11224.33), filed Jul. 6, 2016,entitled “METHODS AND COMPOSITIONS FOR UPREGULATING ENDOGENEOUSANTIOXIDANT SYSTEMS,” and claims priority to U.S. Provisional PatentApplication No. 62/359,113 (Attorney Docket No. 11224.34), filed Jul. 6,2016, entitled “METHODS AND COMPOSITIONS FOR REDUCING DAMAGE ASSOCIATEDWITH OXIDATIVE PHOSPHORYLATION,” and claims the benefit of U.S.Provisional Patent Application No. 62/359,120 (Attorney Docket No.11224.35), filed Jul. 6, 2016, and entitled “METHODS AND COMPOSITIONSFOR SUPPORTING ENDOGENOUS SYSTEMS RELATED TO LIFE SPAN,” the entiredisclosures of which are hereby incorporated by reference.

BACKGROUND

This disclosure pertains to methods and compositions for upregulatingendogenous antioxidant systems. More particularly, it pertains tonutritional supplements configured to upregulate endogenous antioxidantsystems. Additionally, it pertains to methods of manufacturing thesenutritional supplements and methods of administering these nutritionalsupplements. The nutritional supplements can comprise various activeingredients including antioxidant compounds and compounds, whichupregulate endogenous antioxidant systems (e.g., compounds such as alphalipoic acid, resveratrol, curcumin, EGCG, Olivol®, rutin, quercetin, andhesperetin).

Conventional nutritional supplements often comprise nutrients such asvitamins, minerals, dietary elements, fatty acids, and other vitalnutrients. These nutrients often include compounds such as vitamins thatare vital for growth and development, but cannot be produced by thebody. Sometimes nutritional supplements can include exogenousantioxidants such as vitamin C, vitamin E, beta-carotene, and othercarotenoids that provide the body protection against free radicals,provided that the exogenous antioxidants are absorbed and retained bythe body in sufficient concentrations. Because the body cannot producesome of these exogenous antioxidants and because they can be excreted bycertain systems in the body, these exogenous antioxidants must beregularly consumed to provide ongoing protection against free radicals.In addition to systems to utilize exogenous antioxidants, the body alsocomprises endogenous antioxidant systems that can help defend againstfree radical damage. These exogenous antioxidant systems includeendogenous antioxidants such as glutathione and antioxidant enzymes suchas glutathione reductase, glutathione peroxidases,glutathione-S-transferases (GST), superoxide dismutase (SOD), NAD(P)HDehydrogenase, Quinone 1 (NQO-1), Heme Oxygenase 1 (HO-1), andGlutamate-Cysteine Ligase, Catalytic Subunit (GCL).

Although conventional nutritional supplements provide a variety ofbenefits, conventional nutritional supplements are not necessarilywithout their shortcomings. For example, while conventional nutritionalsupplements may provide exogenous antioxidants, conventional nutritionalsupplements do not spur the body to upregulate its own endogenousantioxidant systems. Also, while conventional nutritional supplementsmay provide exogenous antioxidants, the conventional nutritionalsupplements are not configured to provide the long-lasting benefit of anincrease in endogenous antioxidants.

Thus, while some conventional nutritional supplements currently exist,challenges still exist, including those listed above. Accordingly, itwould be an improvement in the art to improve or replace currenttechniques and/or formulations.

BRIEF SUMMARY

The present application discloses compositions and methods forupregulating endogenous antioxidant systems. In some embodiments,compositions may include a nutritional supplement for reducing freeradical damage that comprises an upregulating compound mixtureconfigured to upregulate an endogenous antioxidant system, an exogenousantioxidant mixture; and a mineral mixture. The upregulating compoundmixture may include one or more of alpha lipoic acid, resveratrol,curcumin, EGCG, Olivol®, rutin, quercetin, and hesperetin. The exogenousantioxidant mixture may comprise one or more of mixed carotenoids, betacarotene, retinyl acetate, vitamin C, vitamin D3, vitamin E, mixedtocopherols, vitamin K1, vitamin K2, vitamin B1, vitamin B2, niacin,niacinamide, vitamin B6, folic acid, vitamin B12, biotin, pantothenicacid, inositol, choline bitartrate, coenzyme Q-10, lutein, and lycopene.

In some embodiments, the nutritional supplement may comprise a firstvehicle. In some embodiments the first vehicle may comprise anupregulating compound mixture configured to upregulate an endogenousantioxidant system, and an exogenous antioxidant mixture. In someembodiments, the nutritional supplement may comprise a second vehicle.In some embodiments, the second vehicle may comprise a mineral mixture.The first and second vehicle may comprise a single solid tablet. Theupregulating compound mixture may include one or more of alpha lipoicacid, resveratrol, curcumin, EGCG, Olivol®, rutin, quercetin, andhesperetin. The exogenous antioxidant mixture can comprise one or moreof mixed carotenoids, beta carotene, retinyl acetate, vitamin C, vitaminD3, vitamin E, mixed tocopherols, vitamin K1, vitamin K2, vitamin B1,vitamin B2, niacin, niacinamide, vitamin B6, folic acid, vitamin B12,biotin, pantothenic acid, inositol, choline bitartrate, coenzyme Q-10,lutein, and lycopene.

In some embodiments, the methods for reducing free radical damagecomprise administering a first vehicle, comprising an upregulatingcompound mixture configured to upregulate an endogenous antioxidantsystem and an exogenous antioxidant mixture, and administering a secondvehicle comprising a mineral mixture, where the upregulating compoundmixture is configured to upregulate an endogenous antioxidant system toreduce free radical damage. The endogenous antioxidant system cancomprise a transcription factor such as Nrf2, NF-κB, PPARα, PPARβ/δ, andPPARγ. The transcription factor can promotes transcription of anantioxidant gene such as a Phase II gene, a NQO1 gene, a GCL gene, asulfiredoxin 1 (SRXN1) gene, a thioredoxin reductase 1 (TXNRD1) gene, aHO-1 gene, a GST family gene, and an UDP-glucuronosyltransferase (UGT)family gene.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to describe the manner in which the above-recited and otheradvantages and features of the disclosure can be obtained, a moreparticular description of the disclosure briefly described above will berendered by reference to specific embodiments thereof which areillustrated in the appended drawings. Understanding that these drawingsdepict only typical embodiments of the disclosure and are not thereforeto be considered to be limiting of its scope, the disclosure will bedescribed and explained with additional specificity and detail throughthe use of the accompanying drawings in which:

FIG. 1 illustrates a model of upregulation of endogenous antioxidantsystems;

FIG. 2A illustrates a chemical structure of alpha lipoic acid;

FIG. 2B illustrates a chemical structure of resveratrol;

FIG. 2C illustrates a chemical structure of curcumin;

FIG. 3A illustrates a chemical structure for epigallocatechin gallate(EGCG);

FIG. 3B illustrates a chemical structure of rutin;

FIG. 3C illustrates a chemical structure of quercetin;

FIG. 4 illustrates a chemical structure of hesperetin;

FIG. 5 illustrates fold-activation of PPARα for alpha lipoic acid,resveratrol, curcumin, and EGCG;

FIG. 6 illustrates fold-activation of PPARα for Olivol®, rutin,quercetin, and hesperetin;

FIG. 7 illustrates fold-activation of PPARα for a mixture of alphalipoic acid, resveratrol, curcumin, EGCG, Olivol®, rutin, quercetin, andhesperetin;

FIG. 8 illustrates fold-activation for a known PPARα agonist, GW590735;

FIG. 9 illustrates fold-activation of PPARδ for alpha lipoic acid,resveratrol, curcumin, and EGCG;

FIG. 10 illustrates fold-activation of PPARδ for Olivol®, rutin,quercetin, and hesperetin;

FIG. 11 illustrates fold-activation of PPARδ for a mixture of alphalipoic acid, resveratrol, curcumin, EGCG, Olivol®, rutin, quercetin, andhesperetin;

FIG. 12 illustrates fold-activation for a known PPARδ agonist, GW0742;

FIG. 13 illustrates fold-activation of PPARγ for alpha lipoic acid,resveratrol, curcumin, and EGCG;

FIG. 14 illustrates fold-activation of PPARγ for Olivol®, rutin,quercetin, and hesperetin;

FIG. 15 illustrates fold-activation of PPARγ for a mixture of alphalipoic acid, resveratrol, curcumin, EGCG, Olivol®, rutin, quercetin, andhesperetin;

FIG. 16 illustrates fold-activation for a known PPARγ agonist,rosiglitazone;

FIG. 17 illustrates fold-activation of Nrf2 for alpha lipoic acid,resveratrol, curcumin, and EGCG;

FIG. 18 illustrates fold-activation of Nrf2 for Olivol®, rutin,quercetin, and Hesperetin.

FIG. 19 illustrates fold-activation of Nrf2 for a mixture of alphalipoic acid, resveratrol, curcumin, EGCG, Olivol®, rutin, quercetin, andhesperetin;

FIG. 20 illustrates fold-activation for a known Nrf2 agonist,L-sulphoraphane;

FIG. 21 illustrates percent inhibition of human NF-κB in antagonist modeform for alpha lipoic acid, resveratrol, curcumin, and EGCG;

FIG. 22 illustrates percent inhibition of human NF-κB in antagonist modeform for Olivol®, rutin, quercetin, and hesperetin;

FIG. 23 illustrates percent inhibition of human NF-κB in antagonist modeform for a mixture of alpha lipoic acid, resveratrol, curcumin, EGCG,Olivol®, rutin, quercetin, and hesperetin;

FIG. 24 illustrates an ideal Kaplan-Meier survival curve for a controlpopulation and a population exposed to an ideal test compound;

FIG. 25 illustrates a Kaplan-Meier survival curve for the wormpopulation tested with N356 at 0.1 mg/ml concentration;

FIG. 26 illustrates a Kaplan-Meier survival curve for the wormpopulation tested with N356 at 1.0 mg/ml concentration;

FIG. 27 illustrates a Kaplan-Meier survival curve for the wormpopulation tested with N356 at 10 mg/ml concentration;

FIG. 28 illustrates a dose-dependent extension of lifespan for N356 at0.1 mg/ml, 1.0 mg/ml, and 10 mg/ml compared to DMSO for health spanmeasured as a function of age at 20% mortality;

FIG. 29 illustrates a Kaplan-Meier survival curve for the wormpopulation tested with N357 at the 0.1 mg/ml concentration;

FIG. 30 illustrates a Kaplan-Meier survival curve for the wormpopulation tested with N357 at the 1.0 mg/ml concentration;

FIG. 31 illustrates a Kaplan-Meier survival curve for the wormpopulation tested with N357 at the 10 mg/ml concentration;

FIG. 32 illustrates a dose-dependent extension of lifespan for N357 at0.1 mg/ml, 1.0 mg/ml, and 10 mg/ml compared to DMSO for health spanmeasured as a function of age at 20% mortality;

FIG. 33 illustrates a Kaplan-Meier survival curve for the wormpopulation tested with N108 (resveratrol) at the 0.1 mg/ml and 10 mg/mlconcentrations;

FIG. 34 illustrates a Kaplan-Meier survival curve for the wormpopulation tested with N198 (alpha lipoic acid) at the 0.1 mg/ml and 10mg/ml concentrations;

FIG. 35 illustrates a Kaplan-Meier survival curve for the wormpopulation tested with N347 (hesperidin) at the 0.1 mg/ml and 10 mg/mlconcentrations;

FIG. 36 illustrates a Kaplan-Meier survival curve for the wormpopulation tested with N104 (quercetin) at the 0.1 mg/ml and 10 mg/mlconcentrations;

FIG. 37 illustrates a Kaplan-Meier survival curve for the wormpopulation tested with N346 (rutin hydrate) at the 0.1 mg/ml and 10mg/ml concentrations;

FIG. 38 illustrates fold-activation of Nrf2 by test solutions comparedto control;

FIG. 39 illustrates fold-activation of Nrf2 by alpha lipoic acidcompared to control;

FIG. 40 illustrates fold-activation of Nrf2 by quercetin compared tocontrol; and

FIG. 41 illustrates fold-activation of Nrf2 by resveratrol compared tocontrol.

DETAILED DESCRIPTION

Described herein are nutritional supplement compositions configured toupregulate endogenous antioxidant systems. In some embodiments, themethods and compositions disclosed in the present application includemethods of preparing and compositions of nutritional supplements thatcomprise one or more of an upregulating compound mixture, an exogenousantioxidant mixture, and a mineral mixture. In other embodiments, themethods of preparing nutritional supplements and compositions ofnutritional supplements comprise preparing nutritional supplements thatcomprise an upregulating compound mixture and an exogenous antioxidantmixture in a first part and a mineral mixture in a second part.

In some cases, damage by free radicals in cells of the body is linked toageing and/or other acute and/or chronic diseases. Free radicals caninclude highly reactive atoms or molecules containing unpairedelectrons. Free radicals can cause damage in biological systems when thefree radical captures an electron from another molecule to pair with itsunpaired electron. The molecule from which the electron was capturedthen becomes a free radical itself and seeks to capture another electronfrom another molecule, causing a chain reaction of free radicalproduction.

Often, when a biological molecule loses an electron, it becomes damagedand ceases to function properly which can lead to damage within thecell. Free radicals can also cause cross-linking of biologicalstructures such as cross-linking of DNA. DNA cross-linking may bedamaging to the cell and may lead to ageing and diseases such as cancer.Free radical induced cross-linking may also be related to the formationof wrinkles, the formation of plaque in arteries leading to heartdisease and stroke, and other chronic diseases.

In some cases, mitochondria are thought to be a main target of damage byfree radicals. The production of energy through oxidativephosphorylation in the mitochondria provides the energy that the bodyneeds to live, but also forms free radicals that may cause damage. Whilemost of the free radicals generated during oxidative phosphorylation areneutralized, it is possible in some cases that the generated freeradicals are not neutralized and can cause damage to the mitochondrialDNA and mitochondrial proteins. This damage to the mitochondrial DNA andmitochondrial proteins can lead to decreased mitochondrial efficiency.In some cases, the free radicals generated in the mitochondria can alsoleak into the cell and cause oxidative damage and/or death of the cell.

Some molecules, such as antioxidants, can inhibit the oxidation of othermolecules and thereby neutralize the oxidizing effects of free radicals.Exogenous antioxidants such as thiols or ascorbic acid (vitamin C) canbe found in certain foods and can work to counteract the effects of freeradicals. In some cases, diets containing foods high in these exogenousantioxidants have been shown to improve overall health. Additionally,some nutritional supplements can include exogenous antioxidants such asvitamin C, vitamin E, beta-carotene, and other carotenoids that mayprovide the body some protection against free radicals. In some cases,the exogenous antioxidants must be absorbed and retained by the body insufficient concentration to provide ongoing protection against freeradicals. In other cases, some of these exogenous antioxidants must beregularly consumed because the body cannot produce them and becausecertain systems in the body work to metabolize or excreted them.

Various endogenous antioxidant systems in the body help the body todefend against free radical damage. In some embodiments, theseendogenous antioxidant systems generate endogenous antioxidants such asglutathione that are configured to neutralize free radicals. In otherembodiments, the endogenous antioxidant systems comprise endogenousantioxidant genes and/or enzymes that work to replenish or recharge thesupply of endogenous antioxidants. In yet other embodiments, theendogenous antioxidant systems comprise endogenous antioxidant enzymesthat themselves neutralize free radicals and/or reduce the damage causedby free radicals. These endogenous antioxidant genes and/or enzymes caninclude, but are not limited to, glutathione reductase, glutathioneperoxidases, glutathione-S-transferases (GST), superoxide dismutase(SOD), NAD(P)H Dehydrogenase, Quinone 1 (NQO-1), Heme Oxygenase 1(HO-1), and Glutamate-Cysteine Ligase, Catalytic Subunit (GCL), andproteins encoded by Phase II genes.

In some instances, these endogenous antioxidant systems can beupregulated to provide greater protection against free radical damage.Referring now to FIG. 1, a model of upregulation of an endogenousantioxidant system is illustrated. While various endogenous antioxidantsystems may be upregulated, FIG. 1 illustrates a possible model forupregulation of endogenous antioxidant Phase II genes through atranscription factor, nuclear factor erythroid 2-related factor (Nrf2).The model can include a living cell 10 that comprises an outer membrane12. The cell 10 can also comprise an inner nucleus 14 that is bounded bya nuclear membrane 16. The model can also include an inducer 20 that cansignal upregulation of an endogenous antioxidant system (e.g., Phase IIgenes). The inducer 20 can include any suitable molecule such as asignaling molecule or an upregulating compound that can upregulate anendogenous antioxidant system. In some cases, the inducer 20 can crossthe outer membrane 12 to signal upregulation of an endogenousantioxidant system. In other cases, the inducer 20 can interact with areceptor at the outer membrane 12 to signal upregulation of anendogenous antioxidant system. In yet other cases, the inducer 20 caninteract with one or more signaling molecules and/or signaling complexesto signal upregulation of an endogenous antioxidant system.

In some embodiments, after the inducer 20 crosses the outer membrane 12,it acts to disrupt a complex formed by an inhibitor 30 such as Keap1 anda transcription factor 40 such as Nrf2. Now free of the inhibitor 30,the transcription factor 40 can cross over the nuclear membrane 16 toenter the nucleus 14. In other embodiments, the inducer 20 disrupts thecomplex formed by the inhibitor 30 and the transcription factor 40 bybinding to the inhibitor 30 and allowing the transcription factor 40 tobe freed. In some cases, once the transcription factor 40 enters thenucleus it can interact with and/or activate one or more responseelements 50 such as an Antioxidant Response Element (ARE). The responseelements 50 can then interact to promote transcription of endogenousantioxidant genes 60 (e.g., Phase II genes).

In some embodiments, various transcription factors are involved inupregulating one or more endogenous antioxidant systems. For example,transcription factors involved in upregulating one or more endogenousantioxidant systems can include Nrf2. Nrf2 is a transcription factorthat is encoded in humans by the NFE2L2 gene and that regulatesexpression of antioxidants in response to oxidative damage caused byinjury and inflammation. As described above, Nrf2 can be maintained inthe cytoplasm of the cell under normal conditions and can be degradedfairly quickly. Under oxidative stress conditions, and/or throughinteraction with an inducer, Nrf2 can translocate to the nucleus topromote transcription of antioxidant genes. In some cases, Nrf2 canpromote transcription of various antioxidant genes including Phase IIgenes, NQO1, GCL, sulfiredoxin 1 (SRXN1) and thioredoxin reductase 1(TXNRD1), HO-1, GST family genes, and UDP-glucuronosyltransferase (UGT)family genes.

In some embodiments, transcription factors involved in upregulating oneor more endogenous antioxidant systems can include an NF-κB complex. Insome cases, the NF-κB transcription factor is involved in cellularresponse to free radicals. NF-κB is often referred to as a rapid-actingprimary transcription factor because of its ability to respond quicklyto harmful cellular stimuli. NF-κB responds quickly to harmful cellularstimuli by being watchfully present in the cell in an inhibitor-boundinactive state. Once the cell detects harmful cellular stimuli, NF-κBcan be quickly activated by degrading the bound inhibitor and freeingNF-κB to translocate to the nucleus to promote transcription of certaingenes, including endogenous antioxidant system genes.

In some embodiments, transcription factors involved in upregulating oneor more endogenous antioxidant systems can include the PPAR (peroxisomeproliferator-activated receptors) family of transcription factors. ThePPAR family includes at least the PPARα, PPARβ/δ, and PPARγtranscription factors. Members of the PPAR family are expressed invarious tissues with PPARα expressed at least in liver, kidney, heart,muscle, and adipose tissue and with PPARβ/δ expressed at least in brain,adipose tissue, and skin. PPARγ can be expressed in three differentforms, γ1, γ2, and γ3, with γ1 expressed in most tissues includingheart, muscle, colon, kidney, pancreas, and spleen, with γ2 expressedmainly in adipose tissue, and with γ3 expressed in macrophages, largeintestine, and white adipose tissue. In some cases, PPAR transcriptionfactors can bind with certain receptors (e.g. retinoid X receptors) topromote transcription of certain genes, including endogenous antioxidantgenes 60 (e.g., Phase II genes).

In some embodiments, upregulating compounds include any compound and/ormixture of compounds suitable for upregulating an endogenous antioxidantsystem. For example, upregulating compounds can include any compoundand/or mixture of compounds that act as an inducer to upregulate anendogenous antioxidant system. In some cases, the upregulating compoundscan include any compound that can translocate to the cytoplasm and/ornucleus to upregulate an endogenous antioxidant system. In other cases,the upregulating compounds can include any compound that can interactwith one or more signaling molecules and/or signaling complexes tosignal upregulation of an endogenous antioxidant system. In yet othercases, the upregulating compounds can include any compound that candirectly interact with one or more signaling molecules and/or signalingcomplexes to signal upregulation of an endogenous antioxidant system. Insome cases, the upregulating compounds can include any compound that canindirectly interact with one or more signaling molecules and/orsignaling complexes to signal upregulation of an endogenous antioxidantsystem. In other cases, the upregulating compounds can include anutrient, an herbal supplement, a plant extract, or any other similarcompound.

In some embodiments, upregulating compounds comprise one or more ofalpha lipoic acid, resveratrol, curcumin, EGCG, Olivol®, rutin,quercetin, and hesperetin. For example, the upregulating compounds caninclude lipoic acid. In some cases, lipoic acid can include one or moreof alpha lipoic acid (ALA), racemic alpha lipoic acid, di-hydro alphalipoic acid, R-(+) alpha lipoic acid, S-(−) alpha lipoic acid, R-(+)dihydro alpha lipoic acid, S-(−) dihydro alpha lipoic acid, metal saltsthereof, esters thereof, or combinations thereof. FIG. 2A shows onechemical formula of alpha lipoic acid.

In some embodiments, the upregulating compounds comprise resveratrol ora similar stilbenoid. Resveratrol can include one or more of3,5,4′-trihydroxy-trans-stilbene, 3,4′,5-Stilbenetriol,trans-Resveratrol, (E)-5-(p-Hydroxystyryl)resorcinol, and(E)-5-(4-hydroxystyryl)benzene-1,3-diol. Resveratrol can include thecis-(Z) and/or trans-(E) isomers. Resveratrol can be derived from anysuitable source including plant sources such as grapes or the skin ofgrapes, seeds of muscadine grapes, blueberries, raspberries, mulberries,bilberries, peanuts, Japanese knotweed, and cocoa powder. FIG. 2B showsone chemical formula of resveratrol.

In some embodiments, the upregulating compounds comprise curcumin. Inother embodiments, curcumin comprises one or more of curcumin and anyother suitable curcuminoid, Curcumin can include any suitable tautomericform of curcumin including, but not limited to, a 1,3-diketo form or anenol form. Curcumin can also include any suitable turmeric extract(e.g., desmethoxycurcumin and/or bis-desmethoxycurcumin. FIG. 2C showsone chemical formula of curcumin.

In some embodiments, the upregulating compounds compriseepigallocatechin gallate (EGCG). In other embodiments, the upregulatingcompounds comprise any suitable ester of epigallocatechin and gallicacid. EGCG can also include one or more of[(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl]3,4,5-trihydroxybenzoate,(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-3-yl3,4,5-trihydroxybenzoate, and (−)-Epigallocatechin gallate. EGCG can bederived from any suitable source, include plant sources such as theleaves of white tea, the leaves of green tea, the leaves of black tea,apple skin, plums, onions, hazelnut, pecans, and carob. FIG. 3A showsone chemical formula of EGCG.

In some embodiments, the upregulating compounds comprise rutin. In otherembodiments, rutin comprises one or more of rutoside,quercetin-3-O-rutinoside, phytomelin, birutan, Eldrin, birutan forte,rutin trihydrate, globularicitrin, violaquercetin, and sophorin. Rutincan also include2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-{[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-({[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxy}methyl)oxan-2-yl]oxy}-4H-chromen-4-oneand2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranosyloxy]-4H-chromen-4-one.Rutin can be derived from any suitable source, include plant sourcessuch as Carpobrotus edulis, Ruta graveolens, buckwheat, asparagus, fruitof the fava d'anata tree, fruits and flowers of the pagoda tree,oranges, grapefruits, lemons, limes, mulberry fruit, ash tree fruits,aronia berries, cranberries, and peaches. FIG. 3B shows one chemicalformula of rutin.

In some embodiments, the upregulating compounds comprise Olivol®. Insome cases, Olivol® can comprise any prepared extract of olive fruit. Inother cases, Olivol® can comprise an extract of olive fruit prepared bymethods disclosed in U.S. Pat. No. 6,358,542 and/or U.S. Pat. No.6,361,803, the disclosures of which are hereby incorporated byreference. In yet other cases, Olivol® can be prepared by providingolive pulp by-product of olive oil production, extracting the pulp witha polar aqueous solvent to form an aqueous phase, passing the aqueousphase through a polymeric resin to trap antioxidants on the resin,washing the polymeric resin with polar organic solvent to releaseantioxidants from the resin to produce a solution of antioxidants in thepolar organic solvent. Olivol® can comprise a phenolic antioxidantmixture of tyrosol, hydroxytyrosol, verbacoside, and other relatedcompounds.

In some embodiments, the upregulating compounds comprise quercetin. Insome cases quercetin can include2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one. Quercetin canalso include sophoretin, meletin, quercetine, xanthaurine, quercetol,quercitin, quertine, and flavin meletin. Quercetin can be derived fromany suitable source including plant sources such as capers, radishleaves, carob fiber, dill, cilantro, Hungarian wax pepper, fennelleaves, red onion, radicchio, watercress, buckwheat, kale, chokeberry,cranberry, lingonberry, black plums, cow peas, sweet potato, blueberry,sea buckthorn berry, rowanberry, crowberry, prickly pear cactus fruits,red delicious apples, broccoli, bilberry, black tea, and green tea. FIG.3C shows one chemical formula of quercetin.

In some embodiments, the upregulating compounds comprise hesperetin. Insome cases, hesperetin can include(S)-2,3-Dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one.In other cases, hesperetin can include one or more of hesperidin,hesperetin 7-rutinoside, neohesperidin, 7-neohesperidoside, andhesperetin 7-rhamnoside. Hesperidin can be derived from any suitablesource, including plant sources such as citrus fruits. FIG. 4 shows onechemical formula of hesperetin.

In some embodiments, the methods include methods of preparingnutritional supplements and compositions of nutritional supplements thatcomprise one or more of an upregulating compound mixture, an exogenousantioxidant mixture, and a mineral mixture. In other embodiments, themethods of preparing nutritional supplements and compositions ofnutritional supplements comprise preparing nutritional supplements thatcomprise an upregulating compound mixture and an exogenous antioxidantmixture in a first part and a mineral mixture in a second part. In yetother embodiments, the methods of preparing nutritional supplements andcompositions of nutritional supplements comprise preparing nutritionalsupplements that comprise an upregulating compound mixture in a firstpart, an exogenous antioxidant mixture in a second part, and a mineralmixture in a third part. In some embodiments, the upregulating compoundmixture in a first part and the exogenous antioxidant mixture in asecond part is combined in a single first vehicle and the mineralmixture in a third part is prepared as a single second vehicle.

In some embodiments, the upregulating compound mixture, the exogenousantioxidant mixture, and the mineral mixture are combined in the form ofa single bilayer tablet or capsule. In these embodiments, theupregulating compound mixture and the exogenous antioxidant mixture arecontained within a first part of the tablet or capsule and the mineralmixture is contained in a second part of the tablet or capsule. Thefirst part and the second part can be maintained partially or completelyseparated from each other using any known separation technique. Forexample, these separation techniques can include forming the first partas a homogeneous first layer in the tablet and the second part as ahomogeneous second layer in the table. The contact between the firstlayer and the second layer is minimized because they only contact eachother at the interface between the first and second layers. In otherembodiments, the separation technique includes using one or more of acoating, a film, and an inert layer to separate the first layer andsecond layers.

In some embodiments, a typical tablet shape comprises a caplet which hasabout the shape of a rectangular box. A bi-layer tablet in theseconfigurations can comprise two or more of these boxes sandwichedtogether, with each box comprising a layer. An amount of material thatis in contact at an interface between the layers can be estimated froman amount of material required to coat the entire tablet. The estimationis carried out by determining the amount of material required to coatthe entire tablet and approximating that about half of this amount is anamount needed for the interface between the layers. Because the amountof material required to coat the entire tablet can range from about 1 toabout 5% of the mass of the entire tablet, half of this amount can beapproximated to range between about 0.5% and about 2.5% of the mass ofthe entire tablet. Therefore, about 0.5% and about 2.5% of the mass ofthe entire tablet can be approximated as the amount of material that isin contact at an interface between the layers.

In some embodiments, this separation technique includes forming thefirst part as a first layer in the tablet and the second part as asecond layer in the tablet. Both the first and second layers are formedwith a concentration gradient where one or more of the activeingredients in the bi-layer tablet is concentrated at an exterior of thetablet and minimized at the location at the interface where the twolayers contact each other. In these embodiments, contact between thefirst layer and the second layer is limited to the interface between thefirst and second layers.

In some embodiments, this separation technique includes forming thefirst part as a first layer in the tablet and the second part as asecond layer in the tablet. In these embodiments, the contact betweenthe first layer and the second layer is reduced by providing a barrierbetween the two layers. In some configurations, the barrier can comprisea physical barrier, such as a film of the same material as the capsulethat dissolves on contacting saliva. The physical barrier can have anythickness sufficient to prevent and/or reduce any contact between thetwo layers. In other configurations, the physical barrier can comprise achemical component that prevents the two layers from reacting with eachother. Examples of such chemical components include magnesium carbonate,potassium carbonate and sodium carbonate.

In other embodiments, the first layer is prepared as a first powder andthe second layer is prepared as a second powder. In some cases, thefirst powder and the second powder can be combined. While the first andsecond portions can be mixed, the contact between the two ingredientscan be minimized or eliminated by coating the first and/or secondpowders with a non-reactive layer having a thickness sufficient toprevent any substantial contact and/or reaction between the twoingredients. Examples of non-reactive layers include one or more ofcellulose and food grade wax.

In some embodiments, the first layer is prepared as a first liquid andthe second layer is prepared as a second liquid. A capsule can beprepared that contains a first, inner capsule containing one of thesetwo liquids. The first capsule can be completely contained within asecond, outer capsule that contains the other liquid. Thus, the twoliquids are kept separated from each other by the inner capsule.

In some embodiments, the upregulating compound mixture and the exogenousantioxidant mixture are formulated as a single vehicle (e.g., a singletablet, dosage, or aliquot). While, the upregulating compound mixturecan include any suitable upregulating compound, in some embodiments, theupregulating compound mixture includes one or more of the upregulatingcompounds described above (e.g., alpha lipoic acid, resveratrol,curcumin, EGCG, Olivol®, rutin, quercetin, and hesperetin). In otherembodiments, the upregulating compound mixture includes one or morebioflavonoids (e.g., sulforaphane precursors found in broccoli extracts,sulforaphane, glucoraffnin, and other suitable bioflavonoids). In yetother embodiments, the upregulating compound mixture comprises thecompounds at the concentrations (e.g., mg of active ingredient (AI) and% by weight in mixture) as described in Table 1.

TABLE 1 % by weight Ingredient mg of AI in mixture Alpha Lipoic Acid 2522 Resveratrol 10 9 Curcumin Phytosome Complex 18 16 (Meriva-Bioavailable curcuminoids containing 3.25 mg curcuminoids) Green TeaExtract (standardized to 17.5 15 EGCG) Olivol ® (Olive Fruit Extract)7.5 7 Rutin 10 9 Quercetin 15 13 Hesperidin 10 9

In some embodiments, alpha lipoic acid comprises between about 15 mg toabout 35 mg of AI of the upregulating compound mixture. In otherembodiments, alpha lipoic acid comprises between about 20 mg to about 30mg of AI of the upregulating compound mixture. In yet other embodiments,alpha lipoic acid comprises up to about 5 mg, about 10 mg, about 15 mg,about 20 mg, about 25 mg, about 30 mg, about 35 mg of AI, or anyintermediary value thereof, of the regulating compound mixture.

In some embodiments, alpha lipoic acid comprises between about 15% toabout 35% by weight of the upregulating compound mixture. In otherembodiments, alpha lipoic acid comprises between about 20% to about 25%by weight of the upregulating compound mixture. In yet otherembodiments, alpha lipoic acid comprises up to about 5%, about 10%,about 15%, about 20%, about 25%, about 30%, about 35% by weight of theupregulating mixture, or any intermediary value thereof of theregulating compound mixture.

In some embodiments, resveratrol comprises between about 1 mg to about25 mg of AI of the upregulating compound mixture. In other embodiments,resveratrol comprises between about 5 mg to about 15 mg of AI of theupregulating compound mixture. In yet other embodiments, resveratrolcomprises up to about 1 mg, about 2 mg, about 5 mg, about 8 mg, about 9mg, about 10 mg, about 12 mg, about 15 mg, or any intermediary valuethereof of AI of the regulating compound mixture.

In some embodiments, resveratrol comprises between about 1% to about 25%by weight of the upregulating compound mixture. In other embodiments,resveratrol comprises between about 5% to about 15% by weight of theupregulating compound mixture. In yet other embodiments, resveratrolcomprises up to about 1%, about 2%, about 5%, about 9%, about 10%, about12%, about 15% by weight, or any intermediary value thereof, of theregulating compound mixture. In some embodiments, curcumin comprisesbetween about 10 mg to about 35 mg of AI of the upregulating compoundmixture. In other embodiments, curcumin comprises between about 15 mg toabout 25 mg of AI of the upregulating compound mixture. In yet otherembodiments, curcumin comprises up to about 5 mg, about 10 mg, about 15mg, about 18 mg, about 20 mg, about 22 mg, about 35 mg, about 30 mg, orany intermediary value thereof of AI of the regulating compound mixture.

In some embodiments, curcumin comprises between about 10% to about 35%by weight of the upregulating compound mixture. In other embodiments,curcumin comprises between about 15% to about 25% by weight of theupregulating compound mixture. In yet other embodiments, curcumincomprises up to about 5%, about 10%, about 15%, about 16%, about 20%,about 22%, about 25%, about 30%, about 35% by weight or any intermediaryvalue thereof, of the regulating compound mixture.

In some embodiments, EGCG comprises between about 10 mg to about 35 mgof AI of the upregulating compound mixture. In other embodiments, EGCGcomprises between about 15 mg to about 25 mg of AI of the upregulatingcompound mixture. In yet other embodiments, EGCG comprises up to about 5mg, about 10 mg, about 15 mg, about 18 mg, about 20 mg, about 22 mg,about 35 mg, about 30 mg, or any intermediary value thereof of AI of theregulating compound mixture.

In some embodiments, EGCG comprises between about 10% to about 35% byweight of the upregulating compound mixture. In other embodiments, EGCGcomprises between about 15% to about 25% by weight of the upregulatingcompound mixture. In yet other embodiments, EGCG comprises up to about5%, about 10%, about 15%, about 16%, about 20%, about 22%, about 25%,about 30%, about 35% by weight or any intermediary value thereof, of theregulating compound mixture.

In some embodiments, Olivol® comprises between about 1 mg to about 20 mgof AI of the upregulating compound mixture. In other embodiments,Olivol® comprises between about 5 mg to about 15 mg of AI of theupregulating compound mixture. In yet other embodiments, Olivol®comprises up to about 1 mg, about 2 mg, about 5 mg, about 7.5 mg, about10 mg, about 12 mg, about 15 mg, about 18 mg, about 20 mg, or anyintermediary value thereof, of AI of the regulating compound mixture.

In some embodiments, Olivol® comprises between about 1% to about 15% byweight of the upregulating compound mixture. In other embodiments,Olivol® comprises between about 5% to about 12% by weight of theupregulating compound mixture. In yet other embodiments, Olivol®comprises up to about 1%, about 2%, about 5%, about 7%, about 8%, about10%, about 12%, about 15%, about 18% by weight or any intermediary valuethereof, of the regulating compound mixture.

In some embodiments, rutin comprises between about 1 mg to about 30 mgof AI of the upregulating compound mixture. In other embodiments, rutincomprises between about 5 mg to about 15 mg of AI of the upregulatingcompound mixture. In yet other embodiments, rutin comprises up to about1 mg, about 2 mg, about 5 mg, about 8 mg, about 10 mg, about 12 mg,about 15 mg, about 20 mg, about 25 mg, about 30 mg, or any intermediaryvalue thereof, of AI of the regulating compound mixture.

In some embodiments, rutin comprises between about 1% to about 30% byweight of the upregulating compound mixture. In other embodiments, rutincomprises between about 5% to about 25% by weight of the upregulatingcompound mixture. In yet other embodiments, rutin comprises up to about5%, about 10%, about 13%, about 15%, about 18%, about 20%, about 22%,about 25%, about 30% by weight or any intermediary value thereof, of theregulating compound mixture.

In some embodiments, quercetin comprises between about 1 mg to about 30mg of AI of the upregulating compound mixture. In other embodiments,quercetin comprises between about 10 mg to about 20 mg of AI of theupregulating compound mixture. In yet other embodiments, quercetincomprises up to about 1 mg, about 5 mg, about 10 mg, about 12 mg, about15 mg, about 18 mg, about 20 mg, about 22 mg, about 25 mg, about 30 mg,or any intermediary value thereof, of AI of the regulating compoundmixture.

In some embodiments, quercetin comprises between about 1% to about 30%by weight of the upregulating compound mixture. In other embodiments,quercetin comprises between about 5% to about 25% by weight of theupregulating compound mixture. In yet other embodiments, quercetincomprises up to about 5%, about 10%, about 13%, about 15%, about 18%,about 20%, about 22%, about 25%, about 30% by weight or any intermediaryvalue thereof, of the regulating compound mixture.

While the exogenous antioxidant mixture can comprise any suitableexogenous antioxidant, at least in some embodiments the exogenousantioxidant mixture comprises one or more of mixed carotenoids, betacarotene, retinyl acetate, vitamin C, vitamin D3, vitamin E, mixedtocopherols, vitamin K1, vitamin K2, vitamin B1, vitamin B2, niacin,niacinamide, vitamin B6, folic acid, vitamin B12, biotin, pantothenicacid, inositol, choline bitartrate, coenzyme Q-10, lutein, and lycopene.In other embodiments, the exogenous antioxidant mixture comprises thecompounds at the concentrations (e.g., mg of active ingredient (AI) andinternational unit (IU)) as described in Table 2.

TABLE 2 Ingredient mg of AI IU Mixed Carotenoids * 0.1 100 IU Betacarotene 1.29 2150 IU  Retinyl Acetate 0.258 750 IU Vitamin C (Poly C)** 100 Vitamin D3 (Cholecalciferol) 0.0125 500 IU Vitamin E(d-alpha-tocopheryl succ.) 41.3  50 IU Mixed Tocopherols 20 Vitamin K10.12 Vitamin K2 (menaquinone, MK-7) 0.015 Vitamin B1 (thiamin HCL) 7.5Vitamin B2 (riboflavin) 7.5 Niacin 2.5 Niacinamide 7.5 Vitamin B6(pyridoxine HCL) 8 Folic Acid 0.15 Vitamin B12 (methylcobalamin) 0.05Biotin 0.075 Pantothenic Acid 22.5 Inositol 32 Choline bitartrate 62.5Coenzyme Q-10 3 Lutein 0.15 Lycopene 0.25 * Mixed carotenoids comprisesa mixture of alpha-carotene, beta-carotene, gamma-carotene, and lycopene** Vitamin (Poly C) was a mixture of calcium ascorbate, potassiumascorbate, magnesium ascorbate, and zinc ascorbate.

In some embodiments, the exogenous antioxidant mixture comprisesindividual exogenous antioxidant compounds at any suitableconcentration. For example, mixed carotenoids can comprise between about0.01 and 1 mg of AI or between 1 and about 200 IU, beta carotene cancomprise between about 0.01 and 3 mg of AI or between 1000 and about3000 IU, retinyl acetate can comprise between about 0.01 and 1 mg of AIor between 100 and about 1500 IU, vitamin C can comprise between about10 and 200 mg of AI, vitamin D3 can comprise between about 0.001 and 1mg of AI or between 100 and about 1000 IU, vitamin E can comprisebetween about 10 and 100 mg of AI or between 10 and about 150 IU, mixedtocopherols can comprise between about 1 and 50 mg of AI, vitamin K1 cancomprise between about 0.01 and 1 mg of AI, vitamin K2 can comprisebetween about 0.0001 and 1 mg of AI, vitamin B1 can comprise betweenabout 1 and 20 mg of AI, vitamin B2 can comprise between about 1 and 20mg of AI, niacin can comprise between about 1 and 20 mg of AI,niacinamide can comprise between about 1 and 20 mg of AI, vitamin B6 cancomprise between about 1 and 20 mg of AI, folic acid can comprisebetween about 0.01 and 2 mg of AI, vitamin B12 can comprise betweenabout 0.001 and 2 mg of AI, biotin can comprise between about 0.001 and2 mg of AI, pantothenic acid can comprise between about 1 and 50 mg ofAI, inositol can comprise between about 1 and 100 mg of AI, cholinebitartrate can comprise between about 1 and 200 mg of AI, coenzyme Q-10can comprise between about 0.1 and 20 mg of AI, lutein can comprisebetween about 0.01 and 2 mg of AI, and lycopene can comprise betweenabout 0.01 and 2 mg of AI.

While the mineral mixture can comprise any suitable exogenousantioxidant, at least in some embodiments the mineral mixture comprisesone or more of calcium, calcium citrate, calcium ascorbate, magnesium,magnesium citrate, magnesium ascorbate, iodine, potassium iodine, zinc,zinc citrate, selenium, L-selenomethionine, sodium selenite, copper,copper gluconate, manganese, manganese gluconate, chromium, chromiumpolynicotinate, molybdenum, molybdenum citrate, boron, boron citrate,silicon, calcium silicate, vanadium, vanadium citrate, ultra-traceminerals, and N-acetyl-L-cysteine. In other embodiments, the mineralmixture comprises individual mineral compounds at the concentrations(e.g., mg of active ingredient (AI)) as described in Table 3.

TABLE 3 Ingredient mg of AI Total Calcium 56.25 Calcium Citrate 48.25Calcium Ascorbate^(1,3) 8 Magnesium (citrate)⁴ 50.13 MagnesiumAscorbate^(2,3,4) 6.12 Iodine (potassium iodide) 0.125 Zinc (citrate)5.0 Selenium (L-selenomethionine) 0.0275 Selenium (sodium selenite)0.0225 Copper (gluconate) 0.5 Manganese (gluconate) 0.5 Chromium(polynicotinate) 0.075 Molybdenum (citrate) 0.0125 Boron (citrate) 0.75Silicon (calcium silicate) 1 Vanadium (citrate) 0.01 Ultra-traceMinerals 0.75 N-acetyl L-cysteine 45 ¹Adds an equivalent of 70.9 mg ofAI of vitamin C from calcium ascorbate ²Adds an equivalent of 79.1 mg ofAI of vitamin C from magnesium ascorbate ³Total vitamin C equivalentequals 150 mg of AI ⁴Total magnesium content equals 56.25 mg of AI

In some embodiments, the mineral mixture is configured to provide one ormore cofactors related to upregulating endogenous antioxidant activity.For example, the mineral mixture can provide one or metal ions that actas a cofactor for an antioxidant enzyme and/or one or more endogenousantioxidant systems.

In some embodiments, the mineral mixture comprises individual mineralcompounds at any suitable concentration. For example total calcium cancomprise between about 10 and about 200 mg of AI, calcium citrate cancomprise between about 1 and about 200 mg of AI, calcium ascorbate cancomprise between about 1 and about 200 mg of AI, magnesium citrate cancomprise between about 1 and about 200 mg of AI, magnesium ascorbate cancomprise between about 0.1 and about 20 mg of AI, potassium iodide cancomprise between about 0.001 and about 10 mg of AI, zinc citrate cancomprise between about 0.1 and about 50 mg of AI, L-selenomethionine cancomprise between about 0.001 and about 1 mg of AI, sodium selenite cancomprise between about 0.001 and about 1 mg of AI, copper gluconate cancomprise between about 0.01 and about 10 mg of AI, manganese gluconatecan comprise between about 0.01 and about 10 mg of AI, chromiumpolynicotinate can comprise between about 0.001 and about 1 mg of AI,molybdenum citrate can comprise between about 0.001 and about 1 mg ofAI, boron citrate can comprise between about 0.01 and about 10 mg of AI,calcium silicate can comprise between about 0.1 and about 10 mg of AI,vanadium citrate can comprise between about 0.001 and about 1 mg of AI,ultra-trace minerals can comprise between about 0.01 and about 10 mg ofAI, and N-acetyl-L-cysteine can comprise between about 1 and about 100mg of AI.

In some embodiments, one or more of the upregulating compound mixture,the exogenous antioxidant compound mixtures and the mineral mixture isprepared as a solid formulation. For example, the upregulating compoundmixture and the exogenous antioxidant mixture are formulated as a singlesolid vehicle (e.g., a solid tablet) and the mineral mixture isformulated as a separate single solid vehicle (e.g., a solid tablet). Inother embodiments, one or more of the upregulating compound mixture, theexogenous antioxidant compound mixtures and the mineral mixture isprepared as a liquid formulation (e.g., a liquid capsule). In somecases, the upregulating compound mixture and the exogenous antioxidantmixture are formulated as a single liquid vehicle (e.g., a liquidcapsule) and the mineral mixture is formulated as a separate singleliquid vehicle (e.g., a liquid capsule). In yet other embodiments, oneor more of the upregulating compound mixture, the exogenous antioxidantcompound mixtures and the mineral mixture is prepared as a solidgranular formulation. For example, the upregulating compound mixture andthe exogenous antioxidant mixture are formulated as solid granularvehicle (e.g., a granular filled capsule) and the mineral mixture isformulated as a separate solid granular vehicle (e.g., a granular filledcapsule).

In some embodiments, one or more of the upregulating compound mixture,the exogenous antioxidant compound mixtures and the mineral mixturecomprise any suitable additive. For example, suitable additive caninclude binders, disintegrants, lubricants, flowing agents, flavorings,coatings, and any combination thereof. In some cases, binders caninclude microcrystalline cellulose, modified cellulose (e.g., Klucel),pre-gelatinized starch, or combinations thereof. In other cases,disintegrants can include croscarmellose sodium. Lubricant can includeascorbyl palmitate, vegetable fatty acid, or combinations thereof.Flowing agents can include silicon dioxide. Flavorings can includevanilla extract. In other embodiments, additive may include one or moreof maltodextrin, organic maltodextrin, lecithin, sunflower lecithin,palm olein, organic palm olein, guar gum, and organic guar gum. In yetother embodiments, additives comprise any suitable amount of thenutritional supplement.

The nutritional supplement can be prepared in any suitable form,including but not limited to, tablets, capsules, and powders. Soliddiluents or carriers for the solid forms can be lipids, carbohydrates,proteins, mineral solids (e.g., starch, sucrose, kaolin, dicalciumphosphate, gelatin, acacia, corn syrup, corn starch, talc, and theircombinations), and combinations thereof. Capsules can be formulated withknown diluents and excipients, for example, edible oils, talc, calciumcarbonate, calcium stearate, magnesium stearate, and combinationsthereof. Liquid preparations for oral administration may be prepared inwater or aqueous solutions which advantageously contain suspendingagents, such as for example, sodium carboxymethylcellulose,methylcellulose, acacia, polyvinyl pyrrolidone, polyvinyl alcohol andcombinations thereof.

In some embodiments, the nutritional supplements comprise preservativesin the nature of bactericidal and fungicidal agents including, but notlimited to, parabens, chlorobutanol, benzyl alcohol, phenol, thimerosal,and the like. In some cases, the nutritional supplements can compriseisotonic agents such as sugars or sodium chloride. Carriers and vehiclesinclude vegetable oils, water, ethanol, and polyols, for example,glycerol, propylene glycol, liquid polyethylene glycol, and the like.

The nutritional supplements can be prepared using any known method thatwill manufacture the desired form with the components in the desiredconcentrations. In some embodiments, the ingredients for one of theupregulating compound mixture, the exogenous antioxidant mixture, ormineral mixture are first weighed out and then transferred to a blenderto be mixed. After the respective ingredients have been mixed in theblender, they are transferred to a hopper that feeds a tablet press thatforms compressed tablets. The compressed tablets can be transferred to acoating pan where the coating solution is applied and the tablets aredried. The same process can be repeated for the remaining mixtures.

In some embodiments, the nutritional supplement is administered to ahuman or an animal. While the nutritional supplement can be administeredin any suitable manner, at least in some embodiments, the nutritionalsupplement is configured to be ingested by the human or the animal. Inother embodiments, the method of administration can be adapted to theform of the nutritional supplement. For example, the nutritionalsupplement can be configured in the form of a tablet and/or capsule thatcan be swallowed by a human or an animal. In some cases, the nutritionalsupplement can be configured as a powder and/or a granular solid thatcan be added to a food or a beverage that can be consumed by the humanor the animal. In other cases, the nutritional supplement can beconfigured as a liquid that is encapsulated in a gel capsule that can beswallowed or otherwise ingested. In yet other cases, the nutritionalsupplement can be configured as a liquid that is swallowed or otherwiseingested. In some cases, the nutritional supplement can be configured ina chewable form, such as a gelatin-based chewable dose.

In some embodiments, the nutritional supplement is administered in anysuitable dosage. In other embodiments, the dosage of the nutritionalsupplement is modified based on one or more of an individual's weight,height, age, gender, pregnancy status, breastfeeding status, metabolism,health status, ethnicity, genetics, environment, diet, fitness level,cardiac health, body mass index, and/or lifestyle. In some embodiments,the dosage of the upregulating compound mixture is a daily dose ofbetween about 1 and 6 times the amount listed in Table 1. In otherembodiments, the dosage of the upregulating compound mixture is a dailydosage of about 1, 2, 3, 4, 5, or 6 times the amount listed in Table 1.In yet other embodiments, the dosage of the upregulating compoundmixture is about 4 times the amount listed in Table 1 for an adult. Insome embodiments, the dosage of the upregulating compound mixture isabout 2-3 times the amount listed in Table 1 for an adolescent. In otherembodiments, the dosage of the upregulating compound mixture is about0.5 to 1 times the amount listed in Table 1 for a child.

In some embodiments, the dosage of the endogenous antioxidant compoundmixture is a daily dose of between about 1 and 6 times the amount listedin Table 2. In other embodiments, the dosage of the endogenousantioxidant compound mixture is a daily dosage of about 1, 2, 3, 4, 5,or 6 times the amount listed in Table 2. In yet other embodiments, thedosage of the endogenous antioxidant compound mixture is about 4 timesthe amount listed in Table 2 for an adult. In some embodiments, thedosage of the endogenous antioxidant compound mixture is about 2-3 timesthe amount listed in Table 2 for an adolescent. In other embodiments,the dosage of the endogenous antioxidant compound mixture is about 0.5to 1 times the amount listed in Table 2 for a child.

In some embodiments, the dosage of the mineral compound mixture is adaily dose of between about 1 and 6 times the amount listed in Table 3.In other embodiments, the dosage of the mineral compound mixture is adaily dosage of about 1, 2, 3, 4, 5, or 6 times the amount listed inTable 3. In yet other embodiments, the dosage of the mineral compoundmixture is about 4 times the amount listed in Table 3 for an adult. Insome embodiments, the dosage of the mineral compound mixture is about2-3 times the amount listed in Table 3 for an adolescent. In otherembodiments, the dosage of the mineral compound mixture is about 0.5 to1 times the amount listed in Table 3 for a child.

In some embodiments, the nutritional supplement is administered as asingle daily dose. In other embodiments, the nutritional supplement isadministered as multiple doses within a set period of time (e.g., a 24hour period of time). In yet other embodiments, a single dose is dividedinto aliquots that are administered within a set period of time (e.g., a24 hour period of time). In some embodiments, the nutritional supplementis administered as a single weekly dose. In other embodiments, thenutritional supplement is administered as a single monthly dose.

In some embodiments, the nutritional supplement is daily administeredover a period of days. In other embodiments, the nutritional supplementis administered daily over a period of weeks. In yet other embodiments,the nutritional supplement is administered daily over a period of years.

In some embodiments, the nutritional supplement is administered to ahuman or an animal to reduce and/or prevent free radical damage bysynergistically upregulating endogenous antioxidant systems, providingexogenous antioxidants, and providing minerals. In other embodiments,the nutritional supplement is administered to a human or an animal toupregulate endogenous antioxidant systems within the human or animal. Inyet other embodiments, the nutritional supplement is administered to ahuman or an animal to upregulate endogenous antioxidant systems withinthe human or animal to reduce and/or prevent free radical damage. Insome embodiments, the nutritional supplement is administered to a humanor an animal to reduce and/or prevent damage by free radicals generatedduring oxidative phosphorylation. For example, the nutritionalsupplements can be administered to upregulate Phase II genes to reduceand/or prevent free radical damage. The nutritional supplements can alsobe administered to activate a transcription factor such as Nrf2, NF-κB,PPARα, PPARβ/δ, and/or PPARγ. The nutritional supplements can also beadministered to promote transcription of endogenous antioxidant genesuch as NQO1, GCL, sulfiredoxin 1 (SRXN1) and thioredoxin reductase 1(TXNRD1), HO-1, GST family genes, and UDP-glucuronosyltransferase (UGT)family genes.

Example 1

Various receptor assays were carried out for test compoundscorresponding to ingredients of the nutritional supplement composition.In general, the receptor assays utilized reporter cells that eitherexpressed a native receptor or a receptor hybrid. The receptor hybridswere engineered so that the native N-terminal DNA binding domain (DBD)was replaced with a yeast Gal4 DBD. The reporter cells expressed ahybrid receptor comprising either the native receptor (Nrf2 and NF-κB)or the N-terminal Gal4 DNA binding domain fused to the ligand bindingdomain of the specific human nuclear receptor (PPARα, PPARδ, and PPARγ).The reporter gene (e.g., firefly luciferase) was functionally linked toeither upstream receptor-specific response elements (GRE) or the Gal4upstream activation sequence (UAS). A summary of the receptors, thereporter cells used for each particular receptor assay, and thereference compounds used to confirm performance of the receptor assaysare indicated below in Table 4.

TABLE 4 Refer- Receptor Host ence (gene Recep- Reporter Cell ReferenceAntag- symbol) tor form Vector Line Agonist onist PPARα Gal4 DBD Gal4UAS- CHO GW590735 np (NR1C1) hybrid Luciferase receptor PPARδ Gal4 DBDGal4 UAS- CHO GW0742 np (NR1C2) hybrid Luciferase receptor PPARγ Gal4DBD Gal4 UAS- CHO Rosiglita- np (NR1C3) hybrid Luciferase zone receptorNrf2 Native ARE- CV1 L- np Receptor Luciferase Sulforophane NF-κB NativeNF-κB HEK293 Phorbol ester na NF-κB GRE- (PMA) Luciferase np = assay notperformed na = not available CHO = Chinese hamster ovary cell lineHEK293 = human embryonic kidney 293 cell line CV1 = mammalian CV1 cellline

The test compounds included alpha lipoic acid, resveratrol, curcumin,EGCG, Olivol®, rutin, quercetin, hesperetin, and a mixture of the all ofthe test compounds. A summary of the test compounds are shown below inTable 5.

TABLE 5 Effective Test Raw Actual mass Actual molar molar CompoundMaterial Purity concentration MW concentration concentration Alpha 155mg/ml  74% 114.8 mg/ml  206.32  550 mM 550 μM  lipoic acid Resveratrol 16 mg/ml 47.6%  7.6 mg/ml 228.25 33.3 mM 3.7 μM Curcumin 2.5 100% 2.5mg/ml 368.69 6.79 mM n/a EGCG 20  40%   8 mg/ml 458.372 17.5 mM n/aOlivol ® 20 100%  20 mg/ml 154  129 mM n/a Rutin 30 100%  30 mg/ml610.50 49.1 mM n/a Quercetin 68 100%  68 mg/ml 302.2 22.5 mM 7.5 μMHesperetin 2.5  35% 0.875 mg/ml  610.50 1.43 mM n/a Mixture 7.41 n/a n/an/a n/a n/a

The test compounds were assayed for activity against human PPARα, PPARδ,PPARγ, and Nrf2 in agonist mode. For the agonist assays, separatesuspensions of each of the PPARα, PPARδ, PPARγ, Nrf2 reporter cells wereprepared in cell recovery medium containing 10% charcoal stripped fetalbovine serum. Next, 100 μL aliquots of the PPARα reporter cells weredispensed into each test well of a white 96-well assay plate. Assayplates with PPARδ, PPARγ, and Nrf2 reporter cells were prepared insimilar fashion. Dilutions of the test compounds were serially dilutedusing compound screening medium containing 10% charcoal stripped fetalbovine serum to generate 2×-concentration test compound samples. Controlsolutions of known agonists of the each of the PPARα, PPARδ, PPARγ, andNrf2 receptors were prepared along with a vehicle control. 100 μLaliquots of the 2×-concentration test compound samples, controlsolutions, and vehicle control were dispensed into separate test wellsof each white 96-well assay plate in triplicate. The assay plates wereincubated at 37° C. for 24 h. After incubation, media was removed fromeach test well while leaving behind the receptor cells and 100 μL ofluciferase detection reagent was added to each test well and emittedlight from each test well of the assay plates was detected. The emittedlight from each test well was recorded as relative light units (RLU).

The test compounds were assayed for activity against human NF-κB inantagonist mode. For the agonist assays, a suspension of NF-κB reportercells were prepared in cell recovery medium containing 10% charcoalstripped fetal bovine serum. Next, 100 μL aliquots of the NF-κB reportercells were dispensed into each test well of a white 96-well assay plate.Dilutions of the test compounds were serially diluted using compoundscreening medium containing 10% charcoal stripped fetal bovine serum togenerate 2×-concentration test compound samples. A vehicle control wasprepared. 100 μL aliquots of the 2×-concentration test compound samplesand vehicle control were dispensed into separate test wells of the white96-well assay plate in triplicate. The assay plate was incubated at 37°C. for 24 h. After incubation, media was removed from each test wellwhile leaving behind the receptor cells. The receptor cells were rinsedonce with live cell multiplex buffer, live cell multiplex substrateadded, and the plate incubated at 37° C. for 30 minutes. Afterincubation, fluorescence was measured to determine relative number oflive cell per test well. The live cell multiplex substrate was thenremoved and discarded and 100 μL luciferase detection reagent was addedto each test well and emitted light from each test well of the assayplate was detected. The emitted light from each test well was recordedas relative light units (RLU).

The recorded RLU for each test well was correlated to the respectivenuclear receptor activities by using the RLU of each dilution of eachtest compound, the RLU of the control solutions of known agonists, andthe RLU of the vehicle controls. The fold-activation was determined forthe agonist assays and the percent inhibition and percent live cellswere determined for the antagonist assays. The fold-activation for theagonist assays for each serial dilution of each test compound is shownbelow in Table 6.

TABLE 6 Active PPARα PPARδ PPARγ Nrf2 Ingredient Fold- Fold- Fold- Fold-Test concentration Activa- Activa- Activa- Activa- Compound in μg/mltion tion tion tion Vehicle (DMSO) 0.10% 1.0 1.0 1.0 1.0 Alpha lipoic0.47 1.2 1.4 0.85 1.0 acid 1.42 1.4 1.5 1.1 1.4 4.25 1.3 1.1 1.1 1.412.7 1.2 0.80 1.3 1.5 38.23 1.5 0.52 1.4 1.5 114.70 2.3 0.29 3.4 2.4Resveratrol 0.031 1.3 1.6 1.1 1.5 0.094 1.3 1.1 1.0 1.2 0.28 1.2 1.40.95 1.2 0.85 1.7 1.5 1.3 4.9 2.5 1.8 2.1 3.1 4.0 7.62 1.3 1.5 7.3 3.3Curcumin 0.007 1.3 1.2 1.0 1.6 0.022 1.3 1.2 1.0 1.7 0.067 1.3 1.0 0.951.4 0.20 1.1 1.2 0.96 0.96 0.60 1.0 1.3 0.90 1.0 1.81 0.86 1.0 1.1 1.6EGCG 0.033 1.4 1.3 1.1 1.4 0.098 1.3 1.4 1.0 1.3 0.30 1.4 1.2 1.0 1.20.89 1.2 1.3 0.95 1.2 2.67 1.1 1.1 0.85 1.3 8 0.85 0.48 0.53 0.74Olivol ® 0.8 1.4 0.90 0.78 1.0 0.25 1.5 1.6 1.0 1.4 0.74 1.3 1.1 1.1 1.42.2 1.5 1.6 1.1 1.4 6.7 1.4 1.5 1.0 1.4 20 1.0 0.84 1.0 1.3 Rutin 0.121.1 0.95 0.91 1.4 0.37 1.1 1.0 0.78 1.1 1.11 0.94 1.3 0.86 0.85 3.3 0.991.1 1.0 1.4 10.0 1.0 1.3 1.2 1.4 30 1.0 1.2 1.3 1.5 Quercetin 0.28 1.51.1 1.1 1.5 0.84 1.2 1.0 1.0 1.4 2.5 1.3 1.2 1.0 1.8 7.6 1.1 0.76 1.00.87 23 1.2 0.51 1.5 3.2* 68 0.89 0.26 1.4 4.6* Hesperetin 0.014 1.0 1.01.1 1.6 0.043 1.4 1.1 1.0 1.4 0.130 1.1 1.1 1.1 1.6 0.389 1.2 1.0 1.11.3 1.167 1.0 1.1 1.1 1.5 3.5 0.79 0.89 0.55 0.94 *Greater than 2-foldactivation deemed to be statistically significant

The fold-activation for the agonist assays for each serial dilution ofthe mixture is shown below in Table 7. The undiluted mixture comprised1.22 mg/ml of alpha lipoic acid, 0.49 mg/ml of resveratrol, 0.079 mg/mlof curcumin, 0.34 mg/ml of EGCG, 0.37 mg/ml of Olivol®, 0.49 mg/ml ofrutin, 7.36 mg/ml of quercetin, and 0.49 mg/ml of hesperetin.

TABLE 7 PPARα PPARδ PPARγ Nrf2 Fold- Fold- Fold- Fold- Test Activa-Activa- Activa- Activa- Compound Fold Dilution tion tion tion tionMixture 243,000 1.2 1.3 0.91 1.0 81,000 1.1 1.1 1.0 1.4 27,000 1.3 1.31.0 1.4 9,000 1.2 1.4 1.1 2.0* 3,000 1.5 1.2 1.2 3.5* 1,000 1.5 1.2 2.0*2.7* *Greater than 2-fold activation deemed to be statisticallysignificant

The percent inhibition and percent live cell for the antagonist assaysfor each serial dilution of each test compound is shown below in Table8.

TABLE 8 Active Ingredient Test concentration NF-κB NF-κB Compound inμg/ml % Inhibition % Live Cell Vehicle (DMSO) 0.10%   0.0    0.0 Alphalipoic 0.47   4.4 100 acid 1.42   3.1 100 4.25   −0.70 104 12.7   −6.1102 38.23   5.9 104 114.70 −14  105 Resveratrol 0.031   −9.7 107 0.09 24105 0.28   1.7 100 0.85   −2.6 100 2.54 −50  102 1.81 −28   99 Curcumin0.007 −10  105 0.022   −3.4 105 0.067   −0.29 106 0.20 22 105 0.60 30 99 1.81  51**  97 EGCG 0.033 10 102 0.098 18 101 0.30    0.10 105 0.89  8.4 104 2.67 −15  106 8 −29  107 Olivol ® 0.8 27  99 0.25   −3.8  980.74   −2.2 100 2.2 13  97 6.7 13  99 20 16  98 Rutin 0.12   −3.4 1020.37   2.7 102 1.11 10  99 3.3   −2.3  98 10.0 −11  101 30 −15   97Quercetin 0.28 −12  103 0.84 −6  99 2.5 −12  103 7.6 18  99 23  58**  9768  85**   77*** Hesperetin 0.014   −6.8 101 0.043 −11   98 0.13   −2.5100 0.389   −2.1  99 1.167 14  98 3.5 39  96 **Greater than 2-foldinhibition deemed to be statistically significant ***Possiblecytotoxicity

The fold-activation for the antagonist assays for each serial dilutionof the mixture is shown below in Table 9A. The undiluted mixturecomprised 1.22 mg/ml of alpha lipoic acid, 0.49 mg/ml of resveratrol,0.079 mg/ml of curcumin, 0.34 mg/ml of EGCG, 0.37 mg/ml of Olivol®, 0.49mg/ml of rutin, 7.36 mg/ml of quercetin, and 0.49 mg/ml of Hesperetin.

TABLE 9A Test NF-κB NF-κB Compound Fold Dilution % Inhibition % LiveCell Mixture 243,000 20 96 81,000 11 98 27,000 11 98 9,000 −4.7 96 3,000−40 97 1,000 1.9 97

The results for the receptor assays for human PPARα, PPARδ, PPARγ, andNrf2 in agonist mode, human NF-κB in antagonist mode, and known agonistswere analyzed and are presented graphically as FIGS. 5-23. FIG. 5 showsthe fold-activation of PPARα for alpha lipoic acid, resveratrol,curcumin, and EGCG. FIG. 6 shows the fold-activation of PPARα forOlivol®, rutin, quercetin, and Hesperetin. FIG. 7 shows thefold-activation of PPARα for the mixture. FIG. 8 shows thefold-activation for a known PPARα agonist, GW590735, as the positivecontrol.

FIG. 9 shows the fold-activation of PPARδ for alpha lipoic acid,resveratrol, curcumin, and EGCG. FIG. 10 shows the fold-activation ofPPARδ for Olivol®, rutin, quercetin, and Hesperetin. FIG. 11 shows thefold-activation of PPARδ for the mixture. FIG. 12 shows thefold-activation for a known PPARδ agonist, GW0742, as the positivecontrol.

FIG. 13 shows the fold-activation of PPARγ for alpha lipoic acid,resveratrol, curcumin, and EGCG. FIG. 14 shows the fold-activation ofPPARγ for Olivol®, rutin, quercetin, and Hesperetin. FIG. 15 shows thefold-activation of PPARγ for the mixture. The induction concentration ofalpha lipoic acid and resveratrol was 114.8 μg/mL and 2.54 μg/mLrespectively when they are used alone, but was 1.22 μg/mL and 0.49 μg/mLrespectively in the mixture, indicating the synergistic effect of themixture. FIG. 16 shows the fold-activation for a known PPARγ agonist,rosiglitazone, as the positive control. As shown in the above describedfigures, a strong activity was observed, particularly for PPARγ comparedwith PPARα and PPARδ, either by the ingredients alone or the mixture.PPARγ is known to be a potent regulator of lipid and glucose metabolism,and synthetic PPARγ activators such as TZDs were once used asanti-diabetic drugs. Therefore, such findings have clinical relevance inimproving metabolic health. FIG. 17 shows the fold-activation of Nrf2for alpha lipoic acid, resveratrol, curcumin, and EGCG. FIG. 18 showsthe fold-activation of Nrf2 for Olivol®, rutin, quercetin, andHesperetin. FIG. 19 shows the fold-activation of Nrf2 for the mixture.FIG. 20 shows the fold-activation for a known Nrf2 agonist,L-sulphoraphane.

FIG. 21 shows the percent inhibition of human NF-κB in antagonist modeform for alpha lipoic acid, resveratrol, curcumin, and EGCG. FIG. 22shows the percent inhibition of human NF-κB in antagonist mode form forOlivol®, rutin, quercetin, and Hesperetin. FIG. 23 shows the percentinhibition of human NF-κB in antagonist mode form for the mixture.

An analysis of the data indicated that alpha lipoic acid exhibited verylow-level agonist activity against human PPARα, PPARγ, and Nrf2 at theconcentrations tested. The data also indicated that resveratrolexhibited very low-level agonist activity against human PPARδ and humanNrf2 and mid-level activity against human PPARγ at the concentrationstested. The data also indicated that curcumin exhibited very low-levelantagonist activity against human NF-κB at the concentrations tested.The data also indicated that quercetin exhibited low-level agonistactivity against human Nrf2 and very low-level antagonist activityagainst human NF-κB with some evidence of compounded-inducedcytotoxicity at the concentrations tested. Importantly, the data alsoindicated that the mixture exhibited agonist activity against humanPPARγ and human Nrf2 at a concentration much lower than when they wereused alone.

Example 2

Phenotypic screening with a specialized strain of C. elegans worm wascarried out using two test formulations of the disclosed nutritionalsupplement compositions to assess their effect on epigenetic anti-ageingactivity. A first test formulation comprised the composition asdescribed below in Table 9B and was labeled as “N356.” A second testformulation comprised a combination of alpha lipoic acid, resveratrol,curcumin, EGCG, Olivol®, rutin, quercetin, and Hesperetin as describedbelow in Table 9C and was labeled as “N357.” Each test formulation wastested over a range of concentrations. Dilutions of the testformulations were applied to individual populations of C. elegans andthe lifespan of each population was monitored. Any changes in thelifespan of an individual population compared to a control populationwere recorded and correlated to the respective test formulation (N356 orN357) and the respective dilution (0.1 mg/ml, 1 mg/ml, and 10 mg/ml).

TABLE 9B Active Ingredient: mg: Mixed Carotenoids (alpha, beta, gammacarotene and 0.10 lycopene) Beta carotene (2150 IU tab) 1.29 RetinylAcetate (750 IU) 0.26 Vitamin C (Poly C, Ca, K, Mg and Zn Ascorbates)100.00 Vitamin D3 (Cholecalciferol) [500 IU/tab] 0.0125 Vitamin E(d-alpha-tocopheryl succ. 50 IU) 41.30 Mixed Tocopherols 20.00 VitaminK1 0.12 Vitamin K2 (menaquinone, MK-7) 0.02 Vitamin B1 (thiamin HCL)7.50 Vitamin B2 (riboflavin) 7.50 Niacin 2.50 Niacinamide 7.50 VitaminB6 (pyridoxine HCL) 8.00 Folic Acid 0.15 Vitamin B12 (cyanocobalamin)0.05 Biotin 0.08 Pantothenic Acid 22.50 Alpha Lipoic Acid 25.00Resveratrol 10.00 Curcumin Phytosome Complex (containing 3.25 mg 18.06curcuminoids) Green Tea Extract (standardized to EGCG) 17.50 Olivol ®(Olive Fruit Extract) 7.50 Rutin 10.00 Quercetin Dihydrate 15.00Hesperetin 10.00 Inositol 32.00 Choline bitartrate 62.50 Coenzyme Q-103.00 Lutein 0.15 Lycopene 0.25

TABLE 9C Active Ingredient: mg: Alpha Lipoic Acid 25.00 Resveratrol10.00 Curcumin Phytosome Complex (containing 3.25 mg curcuminoids) 18.06Green Tea Extract (standardized to EGCG) 17.50 Olivol (Olive FruitExtract) 7.50 Rutin 10.00 Quercetin Dihydrate 15.00 Hesperidin 10.00

The N356 and N357 formulations were each prepared as individual 100mg/ml stock solutions in dimethyl sulfoxide (DMSO). Serial dilutions ofeach stock solution were then prepared at 0.1 mg/ml, 1 mg/ml, and 10mg/ml in DMSO. Control solutions of DMSO only were also prepared.Synchronous aged adult populations of C. elegans strain CB5586 wormswere prepared. The CB5586 strain comprises a pharyngeal GFP (greenfluorescent protein) tag and a mutation in the bus-5 gene. Thepharyngeal GFP tag allows for fluorescent images of the worm populationsto be taken. The mutation in the bus-5 gene causes the loss of normalcuticle antigens that permits the cuticle of the worms to becomepermeable to the test formulations and allow direct uptake of the testformulations to avoid interaction of the test formulations withprotective mechanisms of the gut channel. Each serial dilution of eachtest formulation was added to a separate population of the preparedworms. The control solutions were also added to separate populations ofthe prepared worms. The populations of the prepared worms were thenmaintained on standard nematode growth media (NGM) agar plates at 20° C.with sufficient food (Escherichia coli strain OP-50).

Each worm population was then monitored by fluorescent imaging todetermine the number of living and dead worms as a function of time. Thefluorescent imaging was analyzed by software that recognized and countedworms based on their fluorescent intensity compared to backgroundfluorescence. Living worms were automatically distinguished by thesoftware from dead worms based on the degree of movement they exhibitedbetween consecutive fluorescent images. The ability to distinguishbetween living and dead worms allowed the number of living worms andcumulative number of dead worms to be monitored against time. Wormpopulations from a selection of fluorescent images were manually checkedto verify that the software had accurately counted the number of livingand dead worms.

The counts of the living and dead worms as a function of time were thenanalyzed for each of the worm populations and used to prepareKaplan-Meier survival curves and associated statistics for each of theworm populations. FIG. 24 shows an ideal Kaplan-Meier survival curve fora control population and a population exposed to an ideal test compound,“Compound A.” The health span extension is shown as the length of timeafter the last mitotic division that 95% of the test population remainsviable when compared to the control population. The median lifespan isshown as the length of time after the last mitotic division that 50% ofthe test population remains viable when compared to the controlpopulation. The maximum lifespan extension is shown as the length oftime after the last mitotic division that 5% of the test populationremains viable when compared to the control population.

FIG. 25 show a Kaplan-Meier survival curve for the worm populationtested with N356 at the 0.1 mg/ml concentration. FIG. 26 show aKaplan-Meier survival curve for the worm population tested with N356 atthe 1.0 mg/ml concentration. FIG. 27 show a Kaplan-Meier survival curvefor the worm population tested with N356 at the 10 mg/ml concentration.FIG. 28 shows a dose-dependent extension of lifespan for N356 at 0.1mg/ml, 1.0 mg/ml, and 10 mg/ml compared to DMSO for health span measuredas a function of age at 20% mortality.

FIG. 29 show a Kaplan-Meier survival curve for the worm populationtested with N357 at the 0.1 mg/ml concentration. FIG. 30 show aKaplan-Meier survival curve for the worm population tested with N357 atthe 1.0 mg/ml concentration. FIG. 31 show a Kaplan-Meier survival curvefor the worm population tested with N357 at the 10 mg/ml concentration.FIG. 32 shows a dose-dependent extension of lifespan for N357 at 0.1mg/ml, 1.0 mg/ml, and 10 mg/ml compared to DMSO for health span measuredas a function of age at 20% mortality.

The counts of the living and dead worms as a function of time and theKaplan-Meier survival curves and the associated statistics were thenanalyzed for each of the worm populations. The analysis includeddetermining mean and median lifespan. The analysis included anon-parametric test, the Log-Rank test, which compares two survivalfunctions for the overall lifespan assay and provides a reliable p-valuesummarizing the whole experiment. The analysis also included Fisher'sExact Test that calculated the significance of survival functioncomparisons at multiple specific time points throughout the experiment,rather than for the overall lifespan. The results of the analysis areshown below in Tables 10-17. Table 10 shows the restricted mean lifespanfor the N356 treated worm population compared to the DMSO control. Table11 shows the restricted mean lifespan for the N357 treated wormpopulation compared to the DMSO control. Table 12 shows the populationage in days at given percent mortalities for the N356 treated wormpopulation compared to the DMSO control. Table 13 shows the populationage in days at given percent mortalities for the N357 treated wormpopulation compared to the DMSO control. Table 14 shows the Log-RankTest results for the N356 treated worm population compared to the DMSOcontrol. Table 15 shows the Log-Rank Test results for the N356 treatedworm population compared to the DMSO control. Table 16 shows theFisher's Exact Test results for the N356 treated worm populationcompared to the DMSO control. Table 17 shows the Fisher's Exact Testresults for the N357 treated worm population compared to the DMSOcontrol.

TABLE 10 95% Confidence Test Formulation # of Days Standard ErrorInterval DMSO 13.23 0.08 13.06~13.39 N356 at 0.1 mg/ml 15.18 0.1414.90~15.45 N356 at 1.0 mg/ml 10.56 0.36  9.85~11.27 N356 at 10 mg/ml10.87 0.24 10.40~11.34

TABLE 11 95% Confidence Test Formulation # of Days Standard ErrorInterval DMSO 13.23 0.08 13.06~13.39 N357 at 0.1 mg/ml 14.40 0.1514.11~14.70 N357 at 1.0 mg/ml 9.81 0.41  9.00~10.61 N357 at 10 mg/ml11.26 0.26 10.77~11.76

TABLE 12 Test Formulation 25% 50% 75% 90% 100% DMSO 11 14 16 — — N356 at0.1 mg/ml 14 16 — — — N356 at 1.0 mg/ml 7 11 14 18 — N356 at 10 mg/ml 711 16 — —

TABLE 13 Test Formulation 25% 50% 75% 90% 100% DMSO 11 14 16 — — N357 at0.1 mg/ml 11 16 18 — — N357 at 1.0 mg/ml 9 11 14 16 18 N357 at 10 mg/ml9 11 14 18 —

TABLE 14 Condition Chi² P-value Bonferroni P-value DMSO vs. N356 at 0.1mg/ml 105.81 0.0e+00 0.0e+00 DMSO vs. N356 at 1.0 mg/ml 47.47 0.0e+000.0e+00 DMSO vs. N356 at 10 mg/ml 51.94 0.0e+00 0.0e+00

TABLE 15 Condition Chi² P-value Bonferroni P-value DMSO vs. N357 at 0.1mg/ml 45.16 0.0e+00 0.0e+00 DMSO vs. N357 at 1.0 mg/ml 67.03 0.0e+000.0e+00 DMSO vs. N357 at 10 mg/ml 45.17 0.0e+00 0.0e+00

TABLE 16 P-value P-value P-value P-value Condition at 25% at 50% at 75%at 90% DMSO vs. N356 at 0.1 mg/ml 2.7e−12 2.5e−12 1.9e−12 1.5e−12 DMSOvs. N356 at 1.0 mg/ml 5.0e−08 5.0e−08 3.3e−06 0.0093 DMSO vs. N356 at 10mg/ml 2.0e−12 3.5e−11 0.0001 0.0591

TABLE 17 P-value P-value P-value P-value Condition at 25% at 50% at 75%at 90% DMSO vs. N357 at 0.1 mg/ml 6.6e−11 3.0e−11 2.2e−07 9.7e−07 DMSOvs. N357 at 1.0 mg/ml 1.3e−11 1.3e−11 5.2e−08 0.0001 DMSO vs. N357 at 10mg/ml 9.8e−11 9.8e−11 1.2e−06 0.0012

The analysis of the phenotypic screen indicated that both N356 and N357displayed anti-ageing activity. In particular, at the 0.1 mg/mlconcentration, the worm population treated with N356 and the wormpopulation treated with N357 both showed statistically significantincreases in chronological lifespan. Treatment with N356 resulted in astatistically significant improvement in mean life span of about 9%, amaximum improvement in lifespan of about 14.3% at 50% mortality, and amaximum improvement in survival up to about 25% between days 11 and 14(e.g., the mid-lifespan and the late-life span). Likewise, treatmentwith N357 resulted in a statistically significant improvement in meanlifespan of about 9%, a maximum improvement in lifespan of about 14.3%at 50% mortality, and a maximum improvement in survival up to about 16%between days 11 and 14 (mid-lifespan and late-lifespan). The maximumeffect was seen in the combination formulation N356. Both treatmentsalso demonstrated significant improvements to lifespan during the earlystages of the population survival curve that lay between health span andmedian lifespan.

The analysis of the N356 populations indicated that of the 0.1 mg/ml,the 1.0 mg/ml, and the 10 mg/ml concentrations that the 0.1 mg/mlconcentration appeared to be the optimum dose. The N356 0.1 mg/mlconcentration treatment resulted in a statistically significantimprovement in mean life span of 15%, a maximum improvement in lifespanof 27% at 25% mortality, and a maximum improvement in survival up to 25%between days 11 and 14. The Log-Rank test results and the Fisher's Exacttest results showed significance overall for the length of the study andfor each individual time point within the study for the N356 0.1 mg/mlconcentration. The analysis of the N357 populations indicated that ofthe 0.1 mg/ml, the 1.0 mg/ml, and the 10 mg/ml concentrations that the0.1 mg/ml concentration appeared to be the optimum dose. The N357 0.1mg/ml concentration treatment resulted in a statistically significantimprovement in mean life span of 9%, a maximum improvement in lifespanof 14.3% at 50% mortality, and a maximum improvement in survival up to16% between days 11 and 14. The Log-Ran test results and the Fisher'sExact test results showed significance overall for the length of thestudy and for each individual time point within the study for the N3570.1 mg/ml concentration.

Both the N356 and the N357 treatments showed a dose dependent effect asseen in FIGS. 28 and 32. The threshold for a positive effect on lifespanseemed to lie somewhere above 1.0 mg/ml and thus 0.1 mg/ml was acceptedas the optimum dose of those concentrations that were tested. For bothN356 and N357, when the concentration was increased to 1.0 mg/ml, thetreatments caused a decrease in lifespan compared to a control.Similarly, at 10 mg/ml, a decrease in lifespan of the respective wormpopulations was seen for both N356 and N357. It is possible thattreatments with concentrations of N356 and N357 below 0.1 mg/ml may alsoincrease lifespan. This data indicate that the components of theformulation corresponding to antioxidants also increased lifespan abovethe effect produced by the herbal components of the formulation. Thedata indicate that there is a synergistic effect on lifespan from theadministration of the antioxidant components and the herbal components.In some cases, there can be a complimentary effect on lifespan from theadministration of the antioxidant components and the herbal components.

Example 3

Phenotypic screening was carried out as described in EXAMPLE 2 forvarious individual compounds of the N357 formulation. Individualcompounds were assayed to determine any possible individual contributionthat an individual compound may have to overall anti-ageing activity.Solutions of resveratrol, alpha lipoic acid, hesperidin (hesperetin),quercetin, and rutin hydrate were prepared at concentrations of 0.1mg/ml and 10 mg/ml in DMSO. Resveratrol was sourced from Sigma Aldrichat ≧99% HPLC purity and was assigned a sample number of N108.

Alpha lipoic acid was sourced from PureBulk™ USA as a racemic mix of Rand S stereoisomers and was assigned a sample number of N198. Hesperidinwas sourced from Sigma Aldrich at ≧80% purity and was assigned a samplenumber of N347. Quercetin was sourced from Tocris Bioscience at ≧98%HPLC purity and was assigned a sample number of N104. Rutin hydrate wassourced from Sigma Aldrich at ≧94% HPLC purity and was assigned a samplenumber of N346. The counts of the living and dead worms as a function oftime were analyzed for each of the worm populations and used to prepareKaplan-Meier survival curves and associated statistics for each of theworm populations as described above. Log-Rank test and Fisher's Exacttest were also performed.

FIGS. 33 to 37 show Kaplan-Meier survival curves for the wormpopulations treated with resveratrol, alpha lipoic acid, hesperidin,quercetin, and rutin hydrate. FIG. 33 show a Kaplan-Meier survival curvefor the worm population tested with N108 (resveratrol) at the 0.1 mg/mland 10 mg/ml concentrations. FIG. 34 show a Kaplan-Meier survival curvefor the worm population tested with N198 (alpha lipoic acid) at the 0.1mg/ml and 10 mg/ml concentrations. FIG. 35 show a Kaplan-Meier survivalcurve for the worm population tested with N347 (hesperidin) at the 0.1mg/ml and 10 mg/ml concentrations. FIG. 36 show a Kaplan-Meier survivalcurve for the worm population tested with N104 (quercetin) at the 0.1mg/ml and 10 mg/ml concentrations. FIG. 37 show a Kaplan-Meier survivalcurve for the worm population tested with N346 (rutin hydrate) at the0.1 mg/ml and 10 mg/ml concentrations.

The mean and median lifespan for each worm population treated withresveratrol, alpha lipoic acid, hesperidin, quercetin, and rutin hydrateare listed below in Table 18. The age in days at 25%, 50%, 75%, 90%, and100% mortality are listed below in Table 19. The Log-Rank test resultsfor each worm population compared to the DMSO control are shown below inTable 20. The Fisher's Exact Test results for each worm populationcompared to the DMSO control are shown below in Table 21.

TABLE 18 Name Days Std. error 95% C.I. DMSO 13.23 0.08 13.06~13.39 N10810 mg/ml (resveratrol) 15.16 0.15 14.87~15.45 N108 0.1 mg/ml(resveratrol) 13.57 0.19 13.20~13.95 N198 10 mg/ml (alpha lipoic acid)14.85 0.14 14.57~15.13 N198 0.1 mg/ml (alpha lipoic acid) 14.89 0.1814.55~15.24 N347 10 mg/ml (hesperidin) 14.19 0.16 13.87~14.51 N347 0.1mg/ml (hesperidin) 14.55 0.16 14.23~14.86 N104 10 mg/ml (quercetin)12.54 0.27 12.02~13.07 N104 0.1 mg/ml (quercetin) 12.17 0.20 11.78~12.57N346 10 mg/ml (rutin hydrate) 13.43 0.22 13.01~13.86 N346 0.1 mg/ml(rutin hydrate) 12.03 0.21 11.61~12.45

TABLE 19 25% 50% 75% 90% 100% mor- mor- mor- mor- mor- Name talitytality tality tality tality DMSO 11 14 16 — — N108 10 mg/ml(resveratrol) 14 16 18 — — N108 0.1 mg/ml (resveratrol) 11 14 18 — —N198 10 mg/ml (alpha lipoic acid) 14 16 18 — — N198 0.1 mg/ml (alphalipoic acid) 14 16 18 — — N347 10 mg/ml (hesperidin) 11 14 18 — — N3470.1 mg/ml (hesperidin) 11 16 18 — — N104 10 mg/ml (quercetin) 9 14 16 —— N104 0.1 mg/ml (quercetin) 9 11 14 18 — N346 10 mg/ml (rutin hydrate)11 14 16 — — N346 0.1 mg/ml (rutin hydrate) 9 11 14 18 —

TABLE 20 Condition Chi² P-value Bonferroni P-value DMSO vs. N108 at 10mg/ml 86.88 0.0e+00 0.0e+00 DMSO vs. N108 at 0.1 mg/ml 4.59 0.03210.0642 DMSO vs. N198 at 10 mg/ml 60.49 0.0e+00 0.0e+00 DMSO vs. N198 at0.1 mg/ml 55.75 0.0e+00 0.0e+00 DMSO vs. N347 at 10 mg/ml 19.37 1.1e−052.2e−05 DMSO vs. N347 at 0.1 mg/ml 45.04 0.0e+00 0.0e+00 DMSO vs. N104at 10 mg/ml 4.98 0.0256 0.0512 DMSO vs. N104 at 0.1 mg/ml 26.70 2.4e−074.7e−07 DMSO vs. N346 at 10 mg/ml 0.23 0.6322 1.0000 DMSO vs. N346 at0.1 mg/ml 28.00 1.2e−07 2.4e−07

TABLE 21 P-value P-value P-value P-value Condition at 25% at 50% at 75%at 90% DMSO vs. N108 at 10 mg/ml 3.1e−12 2.7e−12 2.9e−12 5.6e−09 DMSOvs. N108 at 0.1 mg/ml 0.0517 0.0108 0.1303 0.1226 DMSO vs. N198 at 10mg/ml 3.2e−12 3.2e−12 9.1e−08 1.1e−05 DMSO vs. N198 at 0.1 mg/ml 2.7e−122.6e−12 2.6e−12 4.4e−06 DMSO vs. N347 at 10 mg/ml 2.6e−08 7.4e−08 0.00280.0313 DMSO vs. N347 at 0.1 mg/ml 1.1e−09 2.5e−12 2.1e−07 8.5e−06 DMSOvs. N104 at 10 mg/ml 0.0330 0.5557 0.0754 0.2111 DMSO vs. N104 at 0.1mg/ml   9e−08 9.8e−08 0.0002 0.0031 DMSO vs. N346 at 10 mg/ml 0.89930.1912 0.7094 1.0000 DMSO vs. N346 at 0.1 mg/ml 2.7e−07 2.7e−07 2.7e−060.0031

The analysis indicated that resveratrol (N108), alpha lipoic acid(N198), and hesperidin (N347) all showed significant positive and dosedependent increase of chronological lifespan. Quercetin (N104) and rutin(N346) appeared to show significant negative effect on chronologicallifespan at certain tested concentrations. FIG. 33 shows thatresveratrol appeared to extend lifespan by about 15% at 10 mg/ml. FIG.34 shows that alpha lipoic acid appeared to extend lifespan by about12.5% at both 0.1 mg/ml and 10 mg/ml. FIG. 35 shows that hesperidinappeared to extend lifespan by about 10% at 0.1 mg/ml. FIG. 36 showsthat quercetin, at least at the concentrations tested and under theconditions tested, appeared to decrease lifespan by about 9% at 0.1mg/ml. FIG. 37 shows that rutin hydrate, at least at the concentrationstested and under the conditions tested, appeared to decrease lifespan byabout 9% at 0.1 mg/ml. The summary of results for the worm populationstreated with resveratrol, alpha lipoic acid, hesperidin, quercetin, andrutin hydrate compared to DMSO control are summarized below in Table 22.

TABLE 22 % Change in Statistically Significant Mean Compound ResultLifespan 0.1 mg/ml (resveratrol) No statistical difference N/A 10 mg/ml(resveratrol) Statistically significant +15% increase 0.1 mg/ml (alphalipoic acid) Statistically significant +12.5%  increase 10 mg/ml (alphalipoic acid) Statistically significant +12.5%  increase 0.1 mg/ml(hesperidin) Statistically significant +10% increase 10 mg/ml(hesperidin) No statistical difference N/A 0.1 mg/ml (quercetin)Statistically significant  −9% decrease 10 mg/ml (quercetin) Nostatistical difference N/A 0.1 mg/ml (rutin hydrate) Statisticallysignificant  −8% decrease 10 mg/ml (rutin hydrate) No statisticaldifference N/A N/A = Not applicable

Example 4

In vitro assays of Nrf2 signaling activity were carried out for each ofalpha lipoic acid, resveratrol, curcumin, EGCG, Olivol®, rutin,quercetin, and hesperetin. The assays were carried out with afluorescent reporter assay that utilized a human retinal epithelial cellline. The assays also included validation by RT-PCR (reversetranscription polymerase chain reaction) of Nrf2 target genes. The humanretinal epithelial cell line was purchased from ATCC (American Type CellCulture, Manassas, Va.) and was configured to function as a fluorescentreporter assay. Test solutions of each of the alpha lipoic acid,resveratrol, curcumin, EGCG, Olivol®, rutin, quercetin, and hesperetinwere prepared by serial dilution. Control solutions were also prepared.Positive control solutions of a known Nrf2 agonist, L-sulphoraphane wasalso prepared. Each test solution was then assayed for Nrf2 signalingactivity using the fluorescent reporter assay and compared to controlassays. The positive control solutions were also tested.

FIG. 38 shows the fold-activation of Nrf2 by each test solution comparedto the control. The minimum inducing concentrations of alpha lipoic acidwas 1.75 of resveratrol was 1.0 and of quercetin was 0.55 FIG. 39 showsthe fold-activation of Nrf2 by alpha lipoic acid at 100 μM and 30 μMagainst a control. The fold activation of 5 μM sulforaphane, as apositive control is also shown. Alpha lipoic acid demonstratedactivation of Nrf2 at both 100 μM and 30 FIG. 40 shows thefold-activation of Nrf2 by quercetin at 10 μM and 1 μM against acontrol. The fold activation of 5 μM sulforaphane, as a positive controlis also shown. Quercetin demonstrated activation of Nrf2 at both 10 μMand 1 with more activation at the 1 μM concentration. FIG. 41 shows thefold-activation of Nrf2 by resveratrol at 10 μM and 1 μM against acontrol. The fold activation of 5 μM sulforaphane, as a positive controlis also shown. Resveratrol demonstrated activation of Nrf2 at both 10 μMand 1 with more activation at the 10 μM concentration.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the disclosure (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the disclosure and does not pose alimitation on the scope of the disclosure otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the disclosure.

It is contemplated that numerical values, as well as other values thatare recited herein are modified by the term “about”, whether expresslystated or inherently derived by the discussion of the presentdisclosure. As used herein, the term “about” defines the numericalboundaries of the modified values so as to include, but not be limitedto, tolerances and values up to, and including the numerical value somodified. That is, numerical values can include the actual value that isexpressly stated, as well as other values that are, or can be, thedecimal, fractional, or other multiple of the actual value indicated,and/or described in the disclosure.

Groupings of alternative elements or embodiments of the disclosuredisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this disclosure are described herein, includingthe best mode known to the inventors for carrying out the disclosure. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the disclosureto be practiced otherwise than specifically described herein.Accordingly, this disclosure includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedelements in all possible variations thereof is encompassed by thedisclosure unless otherwise indicated herein or otherwise clearlycontradicted by context.

In closing, it is to be understood that the embodiments of thedisclosure disclosed herein are illustrative of the principles of thepresent disclosure. Other modifications that may be employed are withinthe scope of the disclosure. Thus, by way of example, but not oflimitation, alternative configurations of the present disclosure may beutilized in accordance with the teachings herein. Accordingly, thepresent disclosure is not limited to that precisely as shown anddescribed.

We claim:
 1. A nutritional supplement for reducing free radical damage,the supplement comprising: an upregulating compound mixture configuredto upregulate an endogenous antioxidant system, comprising at least oneof; an exogenous antioxidant mixture; and a mineral mixture.
 2. Thesupplement of claim 1, wherein the upregulating compound mixturecomprises one or more of alpha lipoic acid, resveratrol, curcumin, andEGCG.
 3. The supplement of claim 1, wherein the upregulating compoundmixture comprises one or more of Olivol®, rutin, quercetin, andhesperetin.
 4. The supplement of claim 1, wherein the exogenousantioxidant mixture comprises one or more of mixed carotenoids, betacarotene, retinyl acetate, vitamin C, vitamin D3, vitamin E, mixedtocopherols, vitamin K1, vitamin K2, vitamin B1, vitamin B2, niacin,niacinamide, vitamin B6, folic acid, vitamin B12, biotin, pantothenicacid, inositol, choline bitartrate, coenzyme Q-10, lutein, and lycopene.5. A nutritional supplement for reducing free radical damage, thesupplement comprising: a first vehicle comprising an upregulatingcompound mixture configured to upregulate an endogenous antioxidantsystem and an exogenous antioxidant mixture; and a second vehiclecomprising a mineral mixture.
 6. The supplement of claim 5, wherein thefirst vehicle is a single solid tablet and wherein the second vehicle isa single solid tablet.
 7. The supplement of claim 5, wherein theupregulating compound mixture comprises one or more of alpha lipoicacid, resveratrol, curcumin, and EGCG.
 8. The supplement of claim 5,wherein the upregulating compound mixture comprises one or more ofOlivol®, rutin, quercetin, and hesperetin.
 9. The supplement of claim 5,wherein the exogenous antioxidant mixture comprises one or more of mixedcarotenoids, beta carotene, retinyl acetate, vitamin C, vitamin D3,vitamin E, mixed tocopherols, vitamin K1, vitamin K2, vitamin B1,vitamin B2, niacin, niacinamide, vitamin B6, folic acid, vitamin B12,biotin, pantothenic acid, inositol, choline bitartrate, coenzyme Q-10,lutein, and lycopene.
 10. A method for manufacturing a nutritionalsupplement for reducing free radical damage, the method comprising:combining a first vehicle comprising an upregulating compound mixtureconfigured to upregulate an endogenous antioxidant system and anexogenous antioxidant mixture, with a second vehicle comprising amineral mixture, wherein the upregulating compound mixture is configuredto upregulate an endogenous antioxidant system to reduce free radicaldamage.
 11. The method of claim 10, wherein the upregulating compoundmixture comprises one or more of alpha lipoic acid, resveratrol,curcumin, and EGCG.
 12. The method of claim 10, wherein the upregulatingcompound mixture comprises one or more of Olivol®, rutin, quercetin, andhesperetin.
 13. The method of claim 10, wherein the exogenousantioxidant mixture comprises one or more of mixed carotenoids, betacarotene, retinyl acetate, vitamin C, vitamin D3, vitamin E, mixedtocopherols, vitamin K1, vitamin K2, vitamin B1, vitamin B2, niacin,niacinamide, vitamin B6, folic acid, vitamin B12, biotin, pantothenicacid, inositol, choline bitartrate, coenzyme Q-10, lutein, and lycopene.14. The method of claim 10, wherein the mineral mixture comprises one ormore of calcium citrate, calcium ascorbate, magnesium citrate, magnesiumascorbate, potassium iodine, zinc citrate, L-selenomethionine, sodiumselenite, copper gluconate, manganese gluconate, chromiumpolynicotinate, molybdenum citrate, boron citrate, calcium silicate,vanadium citrate, ultra-trace minerals, and N-acetyl-L-cysteine
 15. Themethod of claim 10, wherein the endogenous antioxidant system comprisesa transcription factor.
 16. The method of claim 15, wherein thetranscription factor comprises one or more of Nrf2, NF-κB, PPARα,PPARβ/δ, and PPARγ.
 17. The method of claim 16, wherein thetranscription factor promotes transcription of an antioxidant gene. 18.The method of claim 17, wherein the antioxidant gene comprises a PhaseII gene.
 19. The method of claim 17, wherein the antioxidant genecomprise one of more of a NQO1 gene, a GCL gene, a sulfiredoxin 1(SRXN1) gene, a thioredoxin reductase 1 (TXNRD1) gene, a HO-1 gene, aGST family gene, and an UDP-glucuronosyltransferase (UGT) family gene.20. A kit comprising: a nutritional supplement comprising a firstvehicle comprising an upregulating compound mixture configured toupregulate an endogenous antioxidant system and an exogenous antioxidantmixture and a second vehicle comprising a mineral mixture; and acontainer.